Application of CvBv5-3 gene to reduction of immunity of fruit fly and preparation of fruit fly model with low immunity
A technology of low immunity and immunity, applied in the field of molecular biology and genetic engineering, can solve the problem of death of transgenic fruit flies and achieve the effect of enhanced screening
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[0029] 1. Construction of transgenic Drosophila
[0030] According to the sequences at both ends of the open reading frame (ORF, shown in SEQ ID NO.1) of the CvBV5-3 gene and the sequence of the multiple cloning site on the pUASttb vector, combined with the mode of action of homologous recombinase, specific primers were designed. The primer sequences are as follows:
[0031] F: 5'- TTCGTTAACAGATCTGCGGCCGC ATGGTGTTCAAACAAAACCTTAAT-3'
[0032] R: 5'- TCCTCTAGAGGTACCTCTCGAG TTAATCTTTGATAATAATTGGTCC-3';
[0033] Trizol (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from hemocytes of Plutella xylostella after parasitism, and a cDNA library was constructed using the kit SMART RACE cDNA Amplification kit (Clontech, USA). Then, use specific primers F and R to PCR amplify the CvBV5-3 gene fragment with restriction sites from the cDNA library constructed, and use homologous recombinase (ClonExpress II One Step Cloning Kit, Novozyme, Nanjing ) to recombine the fragme...
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